1. Field of the Invention
The present invention relates to a method for purifying teicoplanin A2 which is a glycopeptide antibiotics.
2. Background of the Related Art
The glycopeptide antibiotics show excellent activity against superbacteria which recently comes to the fore as a serious problem to public health and are known to inhibit multiplication of bacteria by disrupting cell wall synthesis of pathogenic gram positive bacteria. Teicoplanin A2, is particularly in a great demand since it is excellent in the medicinal effect but has a low toxicity and a long half life.
Production of teicoplanin A2 mainly depends on purification by fermentation since it is hardly synthesized in a chemical method. One of representative strains which have been used in production of teicoplanin includes Actinoplanes teichomyceticus ATCC 31121 (J. Antibiotics, 276-283, 1978). This strain produces A8327 factor B and A8327 factor C, as well as teicoplanin A1, teicoplanin A2 and teicoplanin A3 through a fermentation process. Therefore, methods which can selectively purify teicoplanin A2 from the fermentation broth of the strain have been continuously developed.
According to the procedure described in U.S. Pat. No. 4,239,751, teicoplanin is isolated from the fermentation broths by filtering it, mixing with a water-insoluble organic solvent at pH 3.5 to dissolve an antibiotic mixture, extracting twice, and condensing the extract at a lower temperature until precipitates of the antibiotic mixture are formed, followed by filtering to recover the precipitates. Then, in order to recover the antibiotic substances remaining in the mycelia, an additional extract using an aqueous solution of acetone is conducted. The precipitate thus obtained contains A8327 factors as well as three types of teicoplanin. Accordingly, the precipitates are subjected to a solvent system to remove A8327 factors and to a chromatography on Sephadex LH 20 to specifically isolate teicoplanin A2.
The above method has defects, related to environmental pollution and an economical burden, in that the final product contains diverse organic solvents accumulated therein due to the use of the organic solvent through several steps and an expensive separation resin such as Sephadex LH 20 should be used to obtain teicoplanin A2, though it enables us to obtain teicoplanin with a high purity.
U.S. Pat. No. 4,542,018 discloses a method for producing pure single factors of teicoplanin A2, teicoplanin A2-1, A2-2, A2-3, A2-4 and A2-5 by reversed phase resin chromatography of teicoplanin A2 obtained by the method according to U.S. Pat. No. 4,239,751.
However, since the method of this patent also uses the expensive reversed phase resin having a small particle size, it cannot be used for the industrial purposes of mass production and still has the problems involved in the method of U.S. Pat. No. 4,239,751.
Meanwhile, U.S. Pat. No. 4,239,751 discloses a method for producing teicoplanin A2, in which a fermentation broth is mixed with a water-miscible solvents such as acetonitrile, acetone, propanol, methylethylketone, etc. without filtration to isolate teicoplanin A2 from the fermentation broth and the resulting solution is condensed, left at a low temperature until precipitates are formed, followed by filtration to recover the precipitates. This method is simpler than that of U.S. Pat. No. 4,239,751, since one step is omitted.
However, the method has disadvantages in that it uses an excessive amount of organic solvents, instead of a filtration process, to separate the filtrate from the mycelia, causing of solvent accumulation and much cost is taken to recover and separate the used solvent mixture.
According to the method disclosed in EP Pat. No. 0479086, in order to increase extraction efficiency of teicoplanin A2, the fermentation broth is adjusted to a predetermined pH, prior to two filtration processes. The filtrate of the fermentation broth is loaded on a polyamide resin and the eluate is extracted with an excessive amount of acetone and left to stand for 3 hours. The supernatant is decanted and the rest is filtered. The resulting cake is washed with acetone to recover teicoplanin A2.
By the method, it is possible to obtain teicoplanin A2 with HPLC purity of 85% at yield of 74.3%. However, such purity is low to be used in production of medicine. Also, it still has the problem of solvent accumulation, since it uses an excessive amount of acetone in diverse steps.
U.S. Pat. No. 4,845,194 discloses a method for producing teicoplanin, in which a cation exchange resin having a cross-linkage of 2% or less is added to the fermentation broth to adsorb teicoplanin to the resin and a 100 mesh sieve is used to separate the mycelia from the resin. Then, the resin is washed with purified water, followed by elution to recover teicoplanin.
However, the cation exchange resin having a cross-linkage of 2% or less which is used for filtration has chemical defects that it can be degraded in oxidation resistance, volume change, exchange capability and physical defects that it can easily break, causing reduction in its life span. Thus, this method has a disadvantage in that the resin should be frequently exchanged, thereby causing an increase in the cost.
Korean Patent Publication No. 2000-0066479 discloses a method for producing teicoplanin A2, in which a fermentation broth is adjusted to pH 11 and centrifuged, and the supernatant is adsorbed onto a synthetic adsorbent resin such as XAD-16, HP-20 and activated carbon or silica gel, eluted with a 50 to 80% methanol solution and purified under reduced pressure to obtain teicoplanin A2 as crude powder. The crude teicoplanin A2 is dissolved in a solution of sodium acetate and purified by sugar affinity chromatography.
According to this method, there may occur problems related to stability of teicoplanin in the vacuum distillation step to remove the methanol solution, after elution from the synthetic adsorbent resin. Also, since it uses an expensive resin and organic solvent, it is not preferably applicable in mass-production.
Therefore, there still remain demands for inventions directed to a method that can be used to mass produce high purity teicoplanin A2 without using an excessive amount of organic solvents or an expensive resin.